Not applicable.
The present invention relates to the field of cationic lipid:DNA complexes (xe2x80x9cCLDCxe2x80x9d). In particular, the present invention relates to lipid:nucleic acid complexes that contain (1) hydrophilic polymer; (2) nucleic acid that has been condensed with organic polycations; and (3) hydrophilic polymer and nucleic acid that has been condensed with organic polycations. The lipid:nucleic acid complexes of this invention show high transfection activity in vivo following intravenous injection and an unexpected increase in shelf life, as determined by in vivo transfection activity.
The present invention further relates to the field of lipidic microparticles, such as liposomes, lipid:DNA complexes, lipid:drug complexes, and microemulsion droplets, attached to proteins. In particular, the invention relates to lipidic microparticles with attached proteins which have been first conjugated to linker molecules having a hydrophilic polymer domain and a hydrophobic domain capable of stable association with the microparticle, or proteins which have been engineered to contain a hydrophilic domain and a lipid moiety permitting stable association with a lipidic microparticle.
Liposomes that consist of amphiphilic cationic molecules are useful non-viral vectors for gene delivery in vitro and in vivo (reviewed in Crystal, Science 270: 404-410 (1995); Blaese et al., Cancer Gene Ther. 2: 291-297 (1995); Behr et al., Bioconjugate Chem. 5: 382-389 (1994); Remy et al., Bioconjugate Chem. 5: 647-654 (1994); and Gao et al., Gene Therapy 2: 710-722 (1995)). In theory, the positively charged liposomes complex to negatively charged nucleic acids via electrostatic interactions to form lipid:nucleic acid complexes. The lipid:nucleic acid complexes have several advantages as gene transfer vectors. Unlike viral vectors, the lipid:nucleic acid complexes can be used to transfer expression cassettes of essentially unlimited size. Since the complexes lack proteins, they may evoke fewer immunogenic and inflammatory responses. Moreover, they cannot replicate or recombine to form an infectious agent and have low integration frequency.
There are a number of publications that demonstrate convincingly that amphiphilic cationic lipids can mediate gene delivery in vivo and in vitro, by showing detectable expression of a reporter gene in culture cells in vitro (Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413-17 (1987); Loeffler et al., Methods in Enzymology 217: 599-618 (1993); Felgner et al., J. Biol. Chem. 269: 2550-2561 (1994)). Because lipid:nucleic acid complexes are on occasion not as efficient as viral vectors for achieving successful gene transfer, much effort has been devoted in finding cationic lipids with increased transfection efficiency (Behr, Bioconjugate Chem. 5: 382-389 (1994); Remy et al., Bioconjugate Chem. 5: 647-654 (1994); Gao et al., Gene Therapy 2: 710-722 (1995)). Lipid:nucleic acid complexes are regarded with enthusiasm as a potentially useful tool for gene therapy.
Several groups have reported the use of amphiphilic cationic lipid:nucleic acid complexes for in vivo transfection both in animals, and in humans (reviewed in Gao et al., Gene Therapy 2: 710-722 (1995); Zhu et al., Science 261: 209-211 (1993); and Thierry et al., Proc. Natl. Acad. Sci. USA 92: 9742-9746 (1995)). However, the technical problems for preparation of complexes that have stable shelf-lives have not been addressed. For example, unlike viral vector preparations, lipid:nucleic acid complexes are unstable in terms of particle size (Behr, Bioconjugate Chem. 5: 382-389 (1994); Remy et al., Bioconjugate Chem. 5: 647-654 (1994); Gao et al., Gene Therapy 2: 710-722 (1995)). It is therefore difficult to obtain homogeneous lipid:nucleic acid complexes with a size distribution suitable for systemic injection. Most preparations of lipid:nucleic acid complexes are metastable. Consequently, these complexes typically must be used within a short period of time ranging from 30 minutes to a few hours. In recent clinical trials using cationic lipids as a carrier for DNA delivery, the two components were mixed at the bed-side and used immediately (Gao et al., Gene Therapy 2: 710-722 (1995)). The structural instability along with the loss of transfection activity of lipid:nucleic acid complex with time have been challenges for the future development of lipid-mediated gene therapy.
Liposomes consisting of amphiphilic cationic molecules are not, of course, the only form of lipidic microparticles and gene therapy is not the only utility for such particles. Lipidic microparticles have also been used for delivery of drugs and other agents to target sites. Targeting of the microparticles is typically achieved through use of a protein attached to the surface of the microparticle, which may, for example be a ligand for cell surface receptor on a cell type of interest. Conversely, the protein may be an antibody which specifically recognizes an antigen on a cell type of interest, such as diseased cells carrying specific markers. Additionally, proteins can be attached for purposes other than targeting. For example, liposomes can contain prodrugs which slowly seep from the liposome into the circulation. An enzyme attached to the liposome can then convert the prodrug into its active form.
Current methods for effecting the attachment of proteins to lipidic microparticles have been of two types. The first type requires introducing a linker molecule bearing an xe2x80x9cactivexe2x80x9d group (one which reacts with a functional group of the protein) into the microparticle composition prior to conjugation of the xe2x80x9cactivatedxe2x80x9d particle with the protein of interest. The disadvantages of methods of this type are: often uncontrollable, incomplete reaction of the protein with the linker; the presence of excess linker on the resulting conjugate, potentially adverse effect of the linker on the ability of the particle, and the inability to incorporate components reactive with the links into the composition of the particle.
The second group of methods employs the steps of (a) attachment of a hydrophobic moiety, such as a hydrocarbon chain, to the protein molecule, (b) dissolving the components of the lipidic microparticle, along with the conjugate of step (a) in the presence of a detergent, and (c) removing the said detergent, effecting the formation of the lipidic particle incorporating the protein conjugate (Torchilin, Immunomethods 4-244-258 (1994); Laukkanen et al., Biochemistry 33:11664-11670 (1994)). These methods have a number of disadvantages, including the imposition of severe limitations on the range of methods by which the particle can be formed, (e.g. the detergent removal technique is required) and by which the drug or other agent can be loaded into the microparticle. Moreover, step (b) requires the dissolution of the microparticle. These methods are therefore unable to attach a protein to a premade particle without first destroying it. The presence of detergent in these methods is unavoidable because without a detergent the hydrophobically modified protein is insoluble in aqueous medium
The xe2x80x9cinsertionxe2x80x9d into liposomes of hydrophilic polymer-lipid linked to a small (5 amino acid) oligopeptide or small oligosaccharide has been reported. (Zalipsky et al., Bioconjugate Chem. 8:111-118 (1997). The peptide and oligosaccharide employed were, however, of a size (molecular weight, 500-3,000 Da) smaller than, or comparable to, the linker itself (molecular weight 2,750 Da). This study therefore provides no guidance for inserting into liposomes or other lipidic microparticles proteins, such as antibodies, or fragments thereof, conjugated to linkers significantly smaller than the protein. In view of the hydrophilic nature of antibodies and other proteins, the art has taught that the larger, protein portion of such a conjugate prevents the hydrophobic linking moiety from stable association with a lipidic microparticle.
The present invention provides a novel method of preparing cationic lipid:nucleic acid complexes that have increased shelf life. In one embodiment, these complexes are prepared by contacting a nucleic acid with an organic polycation, to produce a condensed or partially condensed nucleic acid. The condensed nucleic acid is then combined with an amphiphilic cationic lipid plus a neutral helper lipid such as cholesterol in a nolar ratio from about 2:1 to about 1:2, producing the lipid:nucleic acid complex. Optionally, a hydrophilic polymer is subsequently added to the lipid:nucleic acid complex. Alternatively, the hydrophilic polymer is added to a lipid:nucleic acid complex comprising nucleic acid that has not been not condensed. These lipid:nucleic acid complexes have an increased shelf life, e.g., when stored at 22xc2x0 C. or below, as compared to an identical lipid:nucleic acid complex in which the nucleic acid component has not been contacted with the organic polycation and/or in which the lipid:nucleic acid complex has not been contacted with a hydrophilic polymer.
In a particularly preferred embodiment, the polycation is a polyamine, more preferably a polyamine such as sperrnidine or spermine.
In another preferred embodiment, the lipid:nucleic acid complexes are prepared by combining a nucleic acid with an amphiphilic cationic lipid and then combining the complex thus formed with a hydrophilic polymer. This lipid:nucleic acid complex has an increased shelf life, e.g., when stored at 22xc2x0 C. or below as compared to an identical complex that has not been combined with the hydrophilic polymer.
In one embodiment, the hydrophilic polymer is selected from the group consisting of polyethylene glycol (PEG), polyethylene glycol derivatized with phosphatidyl ethanolamine (PEG-PE), polyethylene glycol derivatized with tween, polyethylene glycol derivatized with distearoylphosphatidylethanolamine (PEG-DSPE), ganglioside GM1 and synthetic polymers.
In one embodiment, the lipid:nucleic acid complex is lyophilized.
In any of the methods and compositions of this invention, the nucleic acid can be virtually any nucleic acid, e.g., a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA), and peptide nucleic acid (PNA) etc., and is most preferably a DNA. In a particularly preferred embodiment, the DNA is an expression cassette capable of expressing a polypeptide in a cell transfected with the lipid:nucleic acid complex.
In one embodiment the lipid:nucleic acid complexes are formed by first forming a liposome, and then combining the formed liposome with condensed or partially condensed nucleic acid to form a lipid;nucleic acid complex. Optionally, the lipid:nucleic acid complex is subsequently contacted with a hydrophilic polymer. The liposomes can alternatively be combined with an uncondensed nucleic acid to form a lipid:nucleic acid complex to which a hydrophilic polymer (e.g., PEG-PE) is later added. A lipid:nucleic acid complex prepared by the combination of nucleic acid and a liposome contacted with a hydrophilic polymer can be subsequently combined with additional hydrophilic polymer. In a preferred embodiment, the lipid and nucleic acid are combined in a ratio ranging from about 1 to about 20, more preferably from about 4 to about 16, and most preferably from about 8 to about 12 nmole lipid:xcexcg nucleic acid. The lipid and hydrophilic polymer are combined in a molar ratio ranging from about 0.1 to about 10%, more preferably from about 0.3 to about 5% and most preferably from about 0.5% to about 2.0% (molar ratio of hydrophilic polymer to cationic lipid of the complex).
It will be appreciated that a targeting moiety (e.g., an antibody or an antibody fragment) can be attached to the lipid and/or liposome before or after formation of the lipid:nucleic acid complex. In a preferred embodiment, the targeting moiety is coupled to the hydrophilic polymer (e.g., PEG), where the targeting moiety/hydrophilic polymer is subsequently added to the lipid:nucleic acid complex. This provides a convenient means for modifying the targeting specificity of an otherwise generic lipid:nucleic acid complex.
In a particularly preferred embodiment, the method of increasing the shelf life of the lipid:nucleic acid complex includes the steps of combining an expression cassette with spermidine or spermine with an amphiphilic cationic lipid plus a helper lipid such as cholesterol, and a Fabxe2x80x2 fragment of an antibody attached to a spacer, e.g., polyethylene glycol, so that the complex has increased shelf life when stored at about 4xc2x0 C.
In one particularly preferred embodiment, the method of increasing the shelf life of the lipid:nucleic acid complex includes the steps of combining an expression cassette with spermidine or spermine with an amphiphilic cationic lipid, and a Fabxe2x80x2 fragment of an antibody attached to a polyethylene glycol derivative. In another particularly preferred embodiment, includes the steps of combining an expression cassette with an amphiphilic cationic lipid, and a Fabxe2x80x2 fragment of an antibody attached to a polyethylene glycol derivative so that the complex has increased shelf life when stored at about 4xc2x0 C.
This invention also provides for a method of transfecting a nucleic acid into a mammalian cell, the method comprising contacting the cell with any one of the lipid:nucleic acid complexes prepared as described above. In one embodiment, the method uses systemic administration of a lipid:nucleic acid complex into a mammal. In a preferred embodiment, the method of transfecting uses intravenous administration of the lipid:nucleic acid complex into a mammal. In a particularly preferred embodiment, the method comprises contacting a specific cell that expresses a ligand that recognizes the Fabxe2x80x2 fragment.
In yet another embodiment, this invention also provides for pharmaceutical composition comprising the lipid:condensed nucleic acid complex described above. The pharmaceutical compositions comprise a therapeutically effective dose of the lipid:nucleic acid complex and a pharmaceutically acceptable carrier or excipient.
In yet another embodiment, the invention also provides a kit for preparing a lipid:nucleic acid complex, the kit comprising a container with a liposome; a container with a nucleic acid; and a container with a hydrophilic polymer, wherein the liposome and the nucleic acid are mixed to form the lipid:nucleic acid complex and wherein the lipid:nucleic acid complex is contacted with the hydrophilic polymer. In a preferred embodiment, the hydrophilic polymer is derivatized with a targeting moiety, preferably an Fabxe2x80x2 fragment. In another preferred embodiment, the nucleic acid is condensed.
This invention also provides for a lipid:condensed nucleic acid complex prepared using the method of increasing shelf life using nucleic acid condensed with an organic polycation, as summarized above.
The invention further provides a method for making lipidic microparticles bearing attached proteins. The method employs proteins which have been conjugated to linker molecules which will stably associate with lipidic microparticles. The invention therefore permits the attachment of proteins to the surface, for example, of lipidic microparticles which have been preformed.